A SUBSET OF 3´ TO 5´ EXORIBONUCLEASES ARE THE PRIMARY ENZYMES RESPONSIBLE FOR THE DEGRADATION OF PGPG IN CYCLIC DI-GMP SIGNALING

Bis-(3´-5´)-cyclic dimeric guanosine monophosphate, or cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates processes such biofilm formation, motility, and virulence. C-di-GMP is synthesized by diguanylate cyclases (DGCs), while phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMPs by previously unidentified enzymes termed PDE-Bs. To identify the PDE-B responsible for pGpG turnover, a screen for pGpG binding proteins in a Vibrio cholerae open reading frame library was conducted to identify potential pGpG binding proteins. This screen led to identification of oligoribonuclease (Orn). Purified Orn binds to pGpG and can cleave pGpG to GMP in vitro. A deletion mutant of orn in Pseudomonas aeruginosa was highly defective in pGpG turnover and accumulated pGpG. Deletion of orn also resulted in accumulation c-di-GMP, likely through pGpG-mediated inhibition of the PDE-As, causing an increase in c-di-GMP-governed auto-aggregation and biofilm. Thus, we found that Orn serves as the primary PDE-B enzyme in P. aeruginosa that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. However, not all bacteria that utilize c-di-GMP signaling also have an ortholog of orn, suggesting that other PDE-Bs must be present. Therefore, we asked whether RNases that cleave small oligoribonucleotides in other species could also act as PDE-Bs. NrnA, NrnB, and NrnC can rapidly degrade pGpG to GMP. Furthermore, they can reduce the elevated aggregation and biofilm formation in P. aeruginosa ∆orn. Together, these results indicate that rather than having a single dedicated PDE-B, different bacteria utilize distinct RNases to cleave pGpG and complete c-di-GMP signaling. The ∆orn strain also has a growth defect, indicating changes in other regulatory processes that could be due to pGpG accumulation, c-di-GMP accumulation, or another effect due to loss of Orn. We sought to investigate the genetic pathways responsible for these growth defect phenotypes by use of a transposon suppressor screen, and also investigated transcriptional changes using RNA-Seq. This work identifies that c-di-GMP degradation intersects with RNA degradation at the point of the Orn and the functionally related RNases.

The Effect of Package Geometry on Moisture Driven Degradation of Polymer Aluminum Capacitors

Polymer aluminum electrolytic capacitors were introduced to provide an alternative to liquid electrolytic capacitors. Polymer electrolytic capacitor electric parameters of capacitance and ESR are less temperature dependent than those of liquid aluminum electrolytic capacitors. Furthermore, the electrical conductivity of the polymer used in these capacitors (poly-3,4ethylenedioxithiophene) is orders of magnitude higher than the electrolytes used in liquid aluminum electrolytic capacitors, resulting in capacitors with much lower equivalent series resistance which are suitable for use in high ripple-current applications. The presence of the moisture-sensitive polymer PEDOT introduces concerns on the reliability of polymer aluminum capacitors in high humidity conditions. Highly accelerated stress testing (or HAST) (110ºC, 85% relative humidity) of polymer aluminum capacitors in which the parts were subjected to unbiased HAST conditions for 700 hours was done to understand the design factors that contribute to the susceptibility to degradation of a polymer aluminum electrolytic capacitor exposed to HAST conditions. A large scale study involving capacitors of different electrical ratings (2.5V – 16V, 100µF – 470 µF), mounting types (surface-mount and through-hole) and manufacturers (6 different manufacturers) was done to determine a relationship between package geometry and reliability in high temperature-humidity conditions. A Geometry-Based HAST test in which the part selection limited variations between capacitor samples to geometric differences only was done to analyze the effect of package geometry on humidity-driven degradation more closely. Raman spectroscopy, x-ray imaging, environmental scanning electron microscopy, and destructive analysis of the capacitors after HAST exposure was done to determine the failure mechanisms of polymer aluminum capacitors under high temperature-humidity conditions.